To examine subpopulations of thymocytes, we have extended the use of flow microfluorometry (FMF) analysis to characterize cell surface and functional parameters of peanut lectin agglutinated cells. In addition to analysis for known antigenic markers, e.g. the Lyt antigens, we have extended the analysis of these cells using fluorescent lectin probes. We have currently undertaken analyses of the functional differentiation of these thymocyte subpopulations for cell-mediated cytotoxicity and antigen, mitogen, or monokine induced proliferation, to assess the extent of functional differentiation determined by the thymus. A contaminant antibody detected by FMF in the antisera from several non-congenic Lyt immunizations has been examined and used to characterize and define a new lymphocyte cell surface antigen Ly 9 which distinguishes lymphocyte populations from all other tissues and cells. Since this marker is expressed on lymphocytic precursors, as well as differentiated lymphocytes, and not on erythrocytic precursors or differentiated red blood cells, it may prove useful for the study of functional lineages of bone marrow precursors.